PROJECT SUMMARY Scaffold-mediated exogenous cells transplantation and growth factors/hormones delivery are two widely-studied alternatives to conventional autologous grafts, the gold standard. For therapeutic translation, however, both approaches encounter various barriers, including safety concerns. Compared to use of exogenous cells/proteins, strategies that promote tissue regeneration by leveraging endogenous cells/signals in situ are more intriguing. Nonetheless, changes in tissue associated with aging (iron accumulation and chronic inflammation) challenge bone regeneration and repair, particularly in older populations. Emerging evidence suggests that the hypoxia-induced factor-1? (HIF-1?) signaling pathway is a central driver of regeneration and angiogenesis. Findings also show sustained activation of HIF-1? by an iron chelator (e.g., Deferoxamine, DFO) is a promising strategy to improve the capacity of regeneration in aged bones where HIF-1? is markedly inhibited by elevated iron levels. Preliminary work by the Sun lab has found that another small molecule, phenamil, shows strong anti-inflammatory ability in addition to playing a powerful role in promoting bone formation by targeting BMP signaling. A locally and sustained drug delivery system and a bio-mimicking scaffold are critical for successful translational application of these promising small molecular drugs. The primary goal of this study is to develop an innovative translational tissue engineering strategy to improve aged large bone regeneration by rejuvenating endogenous signals and reparative cells. Our central hypothesis is that novel bio-mimicking 3D nanofibrous (NF) scaffold-mediated dual- release of small molecules, DFO and phenamil, can improve critical-sized bone defect repair in aged mice through locally: (1) scavenging for detrimental aged-related factors, i.e., excessive iron and inflammatory cytokines; and (2) activating HIF1? and BMP signaling pathways, thereby promoting production of endogenous angiogenic and osteogenic factors, and recruitment of reparative cells (e.g., MSCs, endothelial cells) in situ, for bone regeneration with a primary focus on non-load-bearing bone defects. In Aim 1, we will develop novel, biomimetic 3D NF scaffolds, using our innovative technique of thermally induced nanofiber self-agglomeration (TISA). In Aim 2, we will develop the dual-release system of DFO and phenamil from a 3D NF scaffold to modulate both angiogenesis and osteogenesis in aged cells in vitro. In Aim 3, we will investigate the contribution of local and controlled release of DFO and phenamil from scaffolds for critical- sized cranial bone defect repair in aged mice. The success of this project will establish a novel strategy for challenged bone repair by improving endogenous tissue regeneration.